The central vacuolar system of eukaryotic cells is an interlocking membrane system composed of distinct organelles (including ER, Golgi apparatus, secretory vesicles, secretory granules, plasma membrane, endosomes and lysosomes) and selective transport pathways. Proper functioning of this system is vital for regulating surface expression and secretion of proteins. Using the fungal metabolite brefeldin A (BFA), which dramatically alters both the distribution and flow of membrane through the central vacuolar system, this group has focused on how organelles maintain their structure and identity, and how membrane trafficking between organelles is regulated. These questions have been directed to three different areas: (1) Understanding the biochemical basis of BFA action. We have found that BFA appears to affect the Golgi apparatus by acting on specific membrane targets that regulate the assembly of cytosolic coat proteins onto the cytoplasmic face of this organelle. Altered interaction of the coats with the organelle leads to organelle disassembly, tubule formation and mixing of organelle components within a defined "homotypic" membrane system. The biochemistry of two BFA sensitive coat proteins, beta-COP and ARF are under investigation. (2) The biology and biochemistry of ARF family proteins. The recognition that the activation of the family of small GTP binding proteins called ARF~s is most likely the target for BFA has led us to an extensive study of the biochemistry and cell biology of individual ARF family proteins using in vitro reconstitution of biochemical assays, mutagenesis and expression of wild type and mutant ARF proteins in cells. (3) Characterization of the distribution and flow of membrane within the vacuolar system. We have found that BFA causes the various compartments of the vacuolar system to collapse into new steady states which produce isolated but functioning new organelle units. Traffic within but not between these units continues in the presence of BFA. In summary, our studies with BFA provide new insights about the properties of sets of organelle-specific coat proteins and presents a framework for relating the biochemical regulation of membrane transport to the structure and maintenance of organelles. We have continued studies on the mechanisms of retention of proteins within the early organelles of the vacuolar system.